A lateral flow assay (LFA) system is generally used to detect antigen-antibody reaction. In the LFA system, antibody is immobilized to a membrane in which a fluid sample moves via capillary force, a conjugate pad and a sample pad are connected to an upstream layer of the membrane, and an absorption pad is connected to a downstream layer of the membrane. On the conjugate pad is dried a gold nanoparticle conjugate to which an antibody capable of being selectively bound to a sample substance is immobilized. On the membrane, an antibody selectively reacting with the sample substance and a substance capable of being bound to the antibody immobilized to a gold nanoparticle are immobilized at different locations. The antibody capable of being selectively bound to the sample substance and immobilized to the membrane, and the antibody immobilized to the gold nanoparticle are configured to be bound to the sample substance in a sandwich manner. The absorption pad is composed of a material capable of efficiently absorbing the fluid sample. In the LFA, when a fluid sample solution is dropped onto a sample pad, the antibody-gold nanoparticle conjugate having selectivity with respect to a sample and the antibody immobilized to the membrane are bound in the sandwich shape to form a band at an antibody-immobilized location on the membrane, which can be seen by the naked eye, if the sample is present on the sample pad.
However, a conventional LFA method provides a detection sensitivity of about 1 ng/mL of antigen protein and cannot be applied to a sample which requires higher detection sensitivity. In addition, for more convenient detection, it is necessary to reduce the volume of the sample and a measurement time.
Various membrane sensors are disclosed in the art, for example, “Membrane strip biosensor system for point-of-care testing” (Korean Patent No. 599420); “Composite membrane” (Japanese Patent Publication 2006-507511A); “Electrochemical membrane strip biosensor” (Korean Patent No. 348351); “Method for Determining Concentration of Multiple Analytes in a Single Fluid Sample” (U.S. Pat. No. 7,494,818); “Sensor having membrane and method for manufacturing the same” (Korean Patent No. 591390); “Test Device for Simultaneous Measurement of Multiple Analytes in a Single Sample” (U.S. Patent Publication No. 2005-214161), and the like. However, these publications do not suggest a technique capable of enhancing sensitivity of the sensor through adjustment of the sensitivity using a multi-hole film, detecting multi-component substances at the same time, and reducing a using amount of sample and analyte detection time through vertical injection of the sample.
The inventors of the present invention exerted all possible efforts to develop a high sensitivity membrane biosensor based on techniques, which have not been realized in the related art, and finally developed the present invention based on confirmation that a membrane biosensor prepared by joining a multi-hole film to a receptor-immobilized membrane permits rapid detection of analytes using a small amount of sample.